- Cloning and Expression of the Cathepsin F-like Cysteine Protease Gene in Escherichia coli and Its Characterization
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Han Seung Joo , Kwang Bon Koo , Kyung In Park , Song Hwan Bae , Jong Won Yun , Chung Soon Chang , Jang Won Choi
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J. Microbiol. 2007;45(2):158-167.
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DOI: https://doi.org/2518 [pii]
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In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the 32P-labeled partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3''-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the Cys90, His226, and Asn250 residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3% to 12.5% of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and 35°C, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.
- Enhanced Production of Exopolysaccharides by Fed-batch Culture of Ganoderma resinaceum DG-6556
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Hyun Mi Kim , Soon-Young Paik , Kyung Soo Ra , Kwang Bon Koo , Jong Won Yun , Jang Won Choi
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J. Microbiol. 2006;44(2):233-242.
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DOI: https://doi.org/2360 [pii]
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The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl2 at an initial pH 6.0 and temperature 31°C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass
and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l)
were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose
medium. It may be worth attempting with other mushroom fermentation processes for
enhanced production of mushroom polysaccharides, particularly those with industrial
potential.
- The Influence of the Nucleotide Sequences of Random Shine-Dalgarno and Spacer Region on Bovine Growth Hormone Gene Expression
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Soon-Young Paik , Kyung Soo Ra , Hoon Sik Cho , Kwang Bon Koo , Hyung Suk Baik , Myung Chul Lee , Jong Won Yun , Jang Won Choi
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J. Microbiol. 2006;44(1):64-71.
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DOI: https://doi.org/2335 [pii]
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To investigate the effects of the nucleotide sequences in Shine-Dalgarno (SD) and the spacer region
(SD-ATG) on bovine growth hormone (bGH) gene expression, the expression vectors under
the control of the T7 promoter (pT7-7 vector) were constructed using bGH derivatives (bGH1 &
bGH14) which have different 5''-coding regions and were induced in E. coli BL21(DE3).
Oligonucleotides containing random SD sequences and a spacer region were chemically synthesized
and the distance between the SD region and the initiation codon were fixed to nine bases
in length. The oligonucleotides were annealed and fused to the bGH1 and bGH14 cDNA,
respectively. When the bGH gene was induced with IPTG in E. coli BL21(DE3), some clones containing
only bGH14 cDNA produced considerable levels of bGH in the range of 6.9% to 8.5%
of total cell proteins by SDS-PAGE and Western blot. Otherwise, the bGH was not detected in
any clones with bGH1 cDNA. Accordingly, the nucleotide sequences of SD and the spacer region
affect on bGH expression indicates that the sequences sufficiently destabilize the mRNA secondary
structure of the bGH14 gene. When the free energy was calculated from the transcription initiation
site to the +51 nucleotide of bGH cDNA using a program of nucleic acid folding and hybridization
prediction, the constructs with values below ‒26.3 kcal/mole (toward minus direction)
were not expressed. The constructs with the original sequence of bGH cDNA also did not show
any expression, regardless of the free energy values. Thus, the disruption of the mRNA secondary
structure may be a major factor regulating bGH expression in the translation initiation process.
Accordingly, the first stem-loop among two secondary structures present in the 5''-end region of
the bGH gene should be disrupted for the effective expression of bGH.
- Characterization, Cloning and Expression of the Ferritin Gene from the Korean Polychaete, Periserrula leucophryna
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Byeong Ryong Jeong , Su-Mi Chung , Nam Joo Baek , Kwang Bon Koo , Hyung Suk Baik , Han-Seung Joo , Chung-Soon Chang , Jang Won Choi
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J. Microbiol. 2006;44(1):54-63.
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DOI: https://doi.org/2336 [pii]
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Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial
gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified
by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain
of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin
cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna
cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest
clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino
acid sequences of this novel gene were deposited in the GenBank databases with accession numbers
DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS:
129-653), including a coding nucleotide sequence of 525 bp, a 5''-untranslated region of 128 bp,
and a 3''-noncoding region of 456 bp. The 5''-UTR contains a putative iron responsive element
(IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids
including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the
immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region
encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification
using the designed primers and included the initiation and termination codons; the recombinant
clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE
and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced
and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly
folded and assembled into a homopolymer composed of a single subunit.
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